In-situ PLA (proximity ligation assay) technology was developed by Ulf Landegren et al. (.Söderberg, O., Gullberg, M., Jarvius M. et al, Nature Methods, 2006, 3(12): 995-1000)) and commercialized by Olink Biosciences AB (www.olink.se). In-situ PLA offers extreme signal amplification. Via the use of dual recognition events at the primary level, the specificity is highly increased. This detection principle has been applied to interrogation of fixed tissue/cells (immunohistochemistry-like applications) and to a lesser extent protein arrays.
In the standard design, two affinity-binders (antibodies, affibodies, aptamers etc.) are conjugated to sequence-designed oligonucleotides, the combination denoted proximity probes and used to probe a sample (FIG. 1). If and only if the two affinity-binder reagents bind in proximity of each other a paired set of specialized and sequence matched oligonucleotides (i.e. backbone- and splint oligo) can hybridize to the binder-conjugated oligos and be converted to a circular molecule by ligation reactions. Next, rolling circle amplification (RCA) is used to elongate one of the binder-conjugated oligos. As a result, each correctly bound pair of affinity reagents are converted into localized DNA-spheres (˜1 μm in diameter, also referred to as rolling circle products or RCPs) containing up to a thousand copies of the circular DNA molecule (engineered to contain binding sites for oligonucleotide reporter probes). The detection is accomplished through hybridization of detection oligos complementary to the RCP. Designs requiring three or more oligo-conjugated affinity-reagents binding in proximity for circularization/RCA have also been reported (Protein Diagnostics by Proximity Ligation: Combining Multiple Recognition and DNA Amplification for Improved Protein Analyses, Leuchowius, K-L., et al., Molecular Diagnostics (Second Edition), 2010, Pages 299-306).
There are several possible implementations of the standard design, for example: (1) A single primary antibody in combination with a pair of two oligo-conjugated secondary antibodies. The secondary antibodies specifically recognize two distinct epitopes of the primary antibody (species specific and/or conjugated haptens such as biotin). (2) Two primary antibodies in combination with a pair of two oligo-conjugated secondary antibodies. Primary antibodies need to be of different species origin or conjugated to different haptens. (3) Two oligo-conjugated primary antibodies.
In situ PLA has been used for localized detection of proteins, protein-protein interactions and post-translational modifications in cells and tissues (O. Söderberg, M. Gullberg, M. Jarvius et al., Nat Methods 3 (12), 995-1000 (2006)). Owing to its intrinsic requirement of dual target recognition by pairs of antibodies and the use of rolling circle amplification (RCA) to amplify successful detection events, the assay can attain a very high level of selectivity and sensitivity in the detection of single endogenous proteins or post-translational modifications (M. Jarvius, J. Paulsson, I. Weibrecht et al., Mol Cell Proteomics 6 (9), 1500-1509 (2007); K. J. Leuchowius, M. Jarvius, M. Wickström et al., Mol Cell Proteomics 9 (1), 178-183 (2010)). The same dual recognition also permits detection of protein-protein interactions by targeting two different proteins in a complex.
Expanding the knowledge of the cellular protein interaction networks is vital for a better understanding of several types of diseases, including cancer. Improved methods to study these interaction networks, especially in clinical material, is therefore of great importance both for increasing the knowledge of the underlying disease mechanics, but also for finding new biomarkers for improved disease diagnostics and treatment response prediction. Another context where multiplexed detection of protein-protein interactions could prove of decisive importance is in the field of network pharmacology, where drugs are designed to act on several drug targets simultaneously. The rationale being that as cellular interaction networks are quite robust because of their underlying structure, to perturb these networks and to avoid escape mutations in malignancy, it may prove crucial to target several proteins simultaneously.
There is a need for new methods that can provide information on more than isolated protein interaction events, such as the simultaneous detection of several interactions. Such methods can help monitor the cellular interaction networks, and provide better diagnostics and treatment options.